There are a variety of conditions in humans which are characterized by a high level of bone resorption and by an abnormal balance between bone formation and bone resorption. Among the more common of these are osteoporosis, Paget's disease, and conditions related to the progress of benign and malignant tumors of the bone and metastatic cancers which have been transferred to bone cells from, for example, prostate or breast initial tumors. Other conditions which are associated with changes in collagen metabolism include osteomalacial diseases, rickets, abnormal growth in children, renal osteodystrophy, and a drug-induced osteopenia. Irregularities in bone metabolism are often side effects of thyroid treatments and thyroid conditions per se, such as primary hypothyroidism and thyrotoxicosis as well as Cushing's disease.
It has been recognized that disorders of bone resorption or other conditions characterized by an abnormal balance between bone formation and bone resorption can be detected by altered levels of pyridinium crosslinks in urine (Robins, 1982b; Macek; Black). The crosslinks, which originate from a number of collagen-containing tissues, take the form of compounds containing a central 3-hydroxy pyridinium ring in which the ring nitrogen is derived from the epsilon amino group of either lysine or hydroxylysine (Fujimoto, 1978; Robins, 1982a; Gunja-Smith; Ogawa; Eyre).
The pyridinium crosslink compounds found in urine can be grouped into four general classes: (1) free, native crosslinks having a molecular weight of about 400 daltons (Fujimoto), (2) glycosylated crosslinks and crosslink peptide forms having a molecular weight of between about 550 and 1,000 daltons (Robins, 1983), (3) crosslink peptide forms having a molecular weight between 1,000 and 3,500 daltons (Robins, 1983, 1984, 1987; Henkel; Eyre), and (4) crosslink peptide forms having a molecular weight greater than 3,500 daltons. In normal adult, these forms account for about 38% (1), 40% (2), 15% (3), and 7% (4) of total urinary crosslinks (Daniloff). About 80% of the free crosslinks (group 1 above) in normal adult urine is pyridinoline (Pyd), the ring nitrogen of which derives from hydroxylysine, and about 20% is deoxypyridinoline (Dpd), the ring nitrogen of which derives from lysine. This Pyd/Dpd ratio applies roughly to the other three classes of crosslinks in urine. The higher molecular weight crosslinks can be converted to free crosslinks by acid hydrolysis (Fujimoto, 1978).
Methods for measuring pyridinium crosslinks in urine have been proposed. One of these methods involves the measurement of total hydrolysed Pyd, i.e., Pyd produced by extensive hydrolysis of urinary crosslinks, by quantitating the hydrolysed Pyd peak separated by HPLC (Fujimoto, 1983). The relationship between total hydrolysed Pyd to age was determined by these workers as a ratio to total hydrolysed Pyd/creatinine, where creatinine level is used to normalize crosslink levels to urine concentration and skeletal mass. It was found that this ratio is high in the urine of children, and relatively constant throughout adulthood, increasing slightly in old age. The authors speculate that this may correspond to the loss of bone mass observed in old age.
Studies on the elevated levels of total crosslinks in hydrolyzed urine of patients with rheumatoid arthritis has been suggested as a method to diagnose this disease (Black, 1989). The levels of total hydrolyzed crosslinks for patients with rheumatoid arthritis (expressed as a ratio of total crosslinks measured by HPLC to creatinine) were elevated by a factor of 5 as compared to controls. However, only total hydrolysed Pyd, but not total hydrolysed Dpd, showed a measurable increase.
In a more extensive study using hydrolyzed urines, Seibel et al. showed significant increases in the excretion of Pyd and Dpd crosslinks relative to controls in both rheumatoid and osteoarthritis. The most marked increases for Pyd crosslinks were in patients with rheumatoid arthritis (Seibel).
Assay methods, such as those just noted, which involve HPLC quantitation of crosslinks from hydrolysed samples, or crosslink subfractions from non-hydrolysed samples, are relatively time-consuming and expensive to carry out, and may not be practical for widespread screening or monitoring therapy in bone-metabolism disorders.
Immunoassays have also been proposed for measuring urinary crosslinks. U.S. Pat. No. 4,973,666 discloses an assay for measuring bone resorption by detection in urine of specific pyridinium crosslinks, characterized by specific peptide extensions, associated with bone collagen. Two specific entities having peptide extensions presumed to be associated with bone collagen are described. These are obtained from the urine of patients suffering from Paget's disease, a disease known to involve high rates of bone formation and destruction. The assay relies on immunospecific binding of crosslink compounds containing the specific peptide fragment or extension with an antibody prepared against the crosslink peptide. It is not clear whether and how the concentration of crosslink peptide being assayed relates to total urinary crosslinks.
Robins has described a technique for measuring pyridinoline in urine by use of an antibody specific to hydrolysed Pyd (Robins, 1986). The method has the limitation that the antibody was found to be specific for the hydrolyzed form of Pyd, requiring that the urine sample being tested first be treated under hydrolytic conditions. The hydrolytic treatment increases the time and expense of the assay, and precludes measurements of other native pyridinium crosslinks.
The pyridinium crosslink content of collagen-containing tissues is known to vary in amount and composition according to tissue type. Pyd crosslinks are found in cartilage, bone, intervertebral discs, ligaments, and the aorta. Dpd crosslinks, which are generally less prevalent than Pyd crosslinks, are found in bone, dentine, ligaments, and the aorta. The proportion of Dpd in tissue pyridinium crosslinks appears to be highest in bone, which has a Pyd:Dpd ratio of between about 3:1 and 4:1. Pyridinium crosslinks in cartilage, on the other hand, contain predominantly Pyd.
Ideally, an assay method for assessing bone metabolism, based on pyridinium crosslink levels in urine, should (a) employ a non-hydrolysed urine sample, to avoid the need for acid hydrolysis of the sample, and (b) utilize an antibody reagent to detect native free pyridinium species, for reducing the time and expense of analysis over conventional HPLC-based tests.